Potent and notably non-toxic cell wall biosynthesis inhibitors have dominated treatment regimens for management of bacterial infections in both hospital and outpatient settings for more than fifty years. Recently, however, resistance of bacteria to these antibiotics has reached an alarming level and has begun to erode their once dependable clinical efficacy. Consequently, discovery of new drugs for the cell wall active antibacterial pharmacopoeia is urgently needed.
Cell wall (peptidoglycan) biosynthetic enzymes and their natural substrates can be very useful tools in studies directed to understanding resistance mechanisms at the molecular level or to the search for newer and more effective agents. Although the transpeptidase enzymes are well-known to be targets of the β-lactam antibiotics, studies on the transglycosylation process have been limited primarily due to poor accessibility of the natural transglycosylation substrate, Lipid II and its analogs. Isolation of Lipid II from liquid cultures of bacteria presents several formidable challenges, the most obvious being scale. The level of Lipid II has been estimated to be only 1,000–2,000 molecules per cell in Esherichia coli. Fifteen hundred molecules per cell equates to approximately 2.5 nM in a dense (109 cfu/ml) culture. Although 50 μg of C14-Lipid II has been isolated from Micrococcus luteus, the amount isolated is minuscule, impure and radioactive. (see, i.e., Brotz, H., et al., Antimicrob. Agents Chemother, 42, 154 (1998).) Secondly, complete separation of the miniscule amount of Lipid II from relatively large amounts of cellular lipid and membrane components is very difficult. Hence, there is a need for an ample and sustainable supply of Lipid II to support any meaningful study of the transglycosylation process.
While Lipid II is a useful tool in the study of resistance mechanisms at the molecular level and the search for newer and more effective agents, it has other uses as well. For instance, Lipid II is useful in the quantitation of lysozyme, an enzyme that preferentially hydrolyzes the beta-1,4 glucosidic linkages between N-acetylmuramic acid and N-acetylglucosamine that occur, for example, in the mucopeptide cell wall structure of microorganisms such as Micrococcus lysodeikticus. In addition, certain lantibiotics can be purified by affinity chromatography on a resin containing immobilized Lipid II.